Dicalcium pyrophosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Feb - 23 Jun 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dicalcium pyrophosphate

EC Number:

232-221-5

EC Name:

Dicalcium pyrophosphate

Cas Number:

7790-76-3

Molecular formula:

Ca2O7P2

IUPAC Name:

dicalcium pyrophosphate

Test material form:

solid: particulate/powder

Method

Target gene:

his operon (S. typhimurium), trp operon (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: distilled water was used as solvent/vehicle, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene (10-50 µg/plate)

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: daunomycin (6 µg/plate); ICR 191 acridine (1 µg/plate)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

INITIAL TEST
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

CONFIRMATORY TEST
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies on the test plates compared to the solvent/vehicle control plates

Evaluation criteria:

In general, a 2 or 2.5-fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose response is a further criterion for mutagenic materials.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was seen at all test material concentrations, which did not influence the results of the assay

RANGE-FINDING/SCREENING STUDIES: A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. Starting with the test material concentration of 5 mg/plate (recommended by OECD GL 471) and subsequent serial dilutions (ratio 1:2) following concentrations of test material were applied: 5, 2.5, 1.25, 0.625, 0.313 and 0.156 mg/plate on two bacteria strains (TA 98 and TA 1535) with different mutation type (frameshift and base-pair substitution) with and without metabolic activation.
The highest concentration of that solution was chosen for the main test that caused low cytotoxicity and/or where the precipitate did not interfere with the scoring according to OECD GL 471.

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values were acceptable and compatible with the historical control values.

Any other information on results incl. tables

Test Material concentrations:

Concentration 1: 5 mg/plate

Concentration 2: 2.5 mg/plate

Concentration 3: 1.25 mg/plate

Concentration 4: 0.625 mg/plate

Concentration 5: 0.313 mg/plate

 

 

Table 2:Mean plate counts and standard deviations (SD); strain: TA98

 

Controls/ test substance conc.	Colony count
	TA98
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	18	2	13	2
NK+S9	23	1	20	2
PK	508	36	632	39
PK1+S9	2154	207	2032	116
PK2+S9	139	20	160	11
Conc. 1	18	4	14	2
Conc. 1+S9	21	2	23	4
Conc. 2	17	2	12	4
Conc. 2+S9	25	5	24	7
Conc. 3	20	3	16	8
Conc. 3+S9	30	4	16	3
Conc. 4	15	5	15	1
Conc. 4+S9	25	6	18	1
Conc. 5	22	4	12	3
Conc. 5+S9	27	6	23	3

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (daunomycin)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

 

 

Table 3:Mean plate counts and standard deviations (SD); strain: TA100

 

Controls/ test substance conc.	Colony count
	TA100
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	82	1	87	16
NK+S9	100	10	96	4
PK	508	52	775	202
PK1+S9	1966	109	1783	111
PK2+S9	686	77	724	95
Conc. 1	87	8	84	7
Conc. 1+S9	100	8	85	12
Conc. 2	100	10	79	11
Conc. 2+S9	103	5	93	8
Conc. 3	82	6	80	6
Conc. 3+S9	103	7	88	9
Conc. 4	90	8	82	2
Conc. 4+S9	97	2	94	16
Conc. 5	97	4	79	3
Conc. 5+S9	109	17	89	10

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

 

 

Table 4:Mean plate counts and standard deviations (SD); strain: TA1535

 

Controls/ test substance conc.	Colony count
	TA1535
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	10	3	8	4
NK+S9	9	2	11	4
PK	375	33	312	28
PK1+S9	82	10	47	17
Conc. 1	8	2	8	3
Conc. 1+S9	9	2	9	7
Conc. 2	10	4	8	4
Conc. 2+S9	5	4	10	0
Conc. 3	11	8	14	3
Conc. 3+S9	5	1	8	3
Conc. 4	8	6	9	3
Conc. 4+S9	7	2	6	2
Conc. 5	10	3	11	3
Conc. 5+S9	8	2	9	1

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

 

 

Table 5: Mean plate counts and standard deviations (SD); strain: TA1537

 

Controls/ test substance conc.	Colony count
	TA1537
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	7	2	4	3
NK+S9	7	3	7	4
PK	1635	439	122	15
PK1+S9	144	23	177	29
Conc. 1	7	4	6	3
Conc. 1+S9	6	5	5	1
Conc. 2	7	2	5	2
Conc. 2+S9	6	0	6	1
Conc. 3	6	1	7	1
Conc. 3+S9	10	2	6	1
Conc. 4	8	1	6	4
Conc. 4+S9	8	3	5	1
Conc. 5	6	3	3	2
Conc. 5+S9	10	2	7	5

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (ICR 191 acridine)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

 

 

Table 6:Mean plate counts and standard deviations (SD); strain: WP2uvrA

 

Controls/ test substance conc.	Colony count
	WP2uvrA
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	17	2	27	4
NK+S9	13	2	31	4
PK	1000	30	1350	38
PK1+S9	402	31	357	44
Conc. 1	18	3	35	2
Conc. 1+S9	16	8	35	4
Conc. 2	17	5	35	3
Conc. 2+S9	16	4	29	2
Conc. 3	18	6	25	3
Conc. 3+S9	17	8	32	2
Conc. 4	16	1	26	5
Conc. 4+S9	16	1	36	11
Conc. 5	20	8	34	2
Conc. 5+S9	17	6	35	5

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (4-nitroquinoline-1-oxide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material did not induce reverse gene mutations in Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) or Escherichia coli (WP2 uvrA) tester strains. Therefore, the test material is considered to be not mutagenic in bacteria.